Project Summary/Abstract Identification of sodium taurocholate cotransporting polypeptide (NTCP) as a functional hepatitis B virus (HBV) receptor solved a longstanding puzzle and represented a major breakthrough in the field. Silencing NTCP expression impairs HBV infectivity in primary human hepatocytes and differentiated HepaRG cell line, whereas NTCP overexpression in HepG2 cell line confers HBV susceptibility. Curiously, HepG2/NTCP cells can be efficiently infected by cell culture-derived HBV (cHBV) of genotype D, but not by patient serum-derived HBV (sHBV) of genotype C. In this regard genotype D is unique in lacking 11 residues between the N-terminal myristyolation signal and the NTCP binding motif in the large (L) envelope protein. Another abnormality is that cHBV infected HepG2/NTCP cells, but not HepaRG cells, overproduce hepatitis B e antigen (HBeAg) relative to hepatitis B surface antigen (HBsAg). We found that NTCP overexpression in the absence of infection increased HBV RNAs, replicative DNA, and proteins. In this regard NTCP is a liver-specific transporter of conjugated bile acids, some of which are ligands and activators of transcription factor FXR?. FXR? stimulates transcription of the 3.5-kb HBV RNA, which drives genome replication and HBeAg but not HBsAg expression. In this R21 grant application we will clarify both the receptor function of NTCP and its transcriptional impact. In Aim 1 we will compare cHBV infectivity between genotype D and non-D genotypes in HepG2/NTCP cells, and establish the impact of the 11 residues in L protein on cHBV infectivity through site-directed mutagenesis. Aim 2 will employ a HepG2 cell line with inducible NTCP expression to estimate the post-entry effect of NTCP on HBV RNA, DNA, and HBsAg/HBeAg ratio following cHBV infection, and on HBV DNA replication and protein production driven by an integrated HBV genome. Aim 3 will verify whether NTCP promotes HBV RNA transcription through bile acids and transcription factor FXR?. This will be achieved by depleting bile acids from culture medium, by NTCP mutants impaired in bile acid transport, and by FXR? silencing. The proposed studies should clarify whether relative to HepaRG cells, HepG2/NTCP cells are poorly susceptible to sHBV infection, or to non-D HBV genotypes. This should spur the search for additional host factors for efficient HBV infection in cell culture. A role of NTCP in up regulating HBV RNA transcription, an early step in viral life cycle, makes it an attractive therapeutic target for chronic HBV carriers. !